This was an open-label, phase Ib lactation study designed to assess the concentration of allopregnanolone in the breast milk of healthy adult lactating women during and after brexanolone intravenous infusion. The study enrolled 12 non-smoking participants aged 18–45 years who were ≤ 6 months postpartum and lactating. Participants agreed to temporarily discontinue breastfeeding until day 7 after initiation of brexanolone infusion. Participants agreed to maximally pump breast milk (at least every 12 h) from day 1 until day 7. Participants with renal failure, fulminant hepatic failure, anemia, known allergy to progesterone or allopregnanolone, or exposure to other investigational study drugs or devices within 30 days prior to screening were excluded.

The study was conducted at six clinical sites in the United States. The study protocol was reviewed and approved by an Independent Ethics Committee and/or Institutional Review Board (Schulman Associates Institutional Review Board, Inc; Cincinnati, OH, USA), and the study was conducted in accordance with the Declaration of Helsinki, the International Conference for Harmonisation, and Good Clinical Practice guidelines. Written informed consent was obtained from all participants before starting any study procedure.

Participants received a single 60-h continuous infusion of brexanolone, with a dosing regimen as follows: 30 μg/kg/h (BRX30; 0–4 h), 60 μg/kg/h (BRX60; 4–24 h), BRX90 (24–52 h), BRX60 (52–56 h), and BRX30 (56–60 h) [23]. Participants were discharged after completion of hour 72 safety assessments (see below) and were followed up with an outpatient visit on day 7. Blood samples for analysis of plasma allopregnanolone were collected pre-infusion at day 1, at pre-defined time points (hours 12, 24, 36, 48, 56, 60, 61, 62, and 64) until 72 h after the start of infusion, and at day 7. PK blood draws during the treatment period had a window of ± 10 min. In the event of an unplanned dose adjustment, the unscheduled PK sample was collected just prior to the infusion rate change. Breast milk samples were collected pre-infusion; at least every 12 h between hour 0 and hour 72; and at days 4, 5, 6, and 7. The collected plasma and breast milk samples were kept frozen until analyzed.

The primary endpoint was allopregnanolone levels in breast milk after a 60-h infusion of brexanolone in healthy lactating women. Secondary endpoints were allopregnanolone levels in plasma after a 60-h brexanolone infusion and the safety and tolerability of a 60-h brexanolone infusion in healthy lactating women.

Plasma samples were collected for PK analyses at predefined time points. Breast milk samples were collected ad libitum and retained for analysis. Allopregnanolone levels in plasma and breast milk were determined using a validated high-performance liquid chromatography–mass spectroscopy (LC–MS) method. The lower limit of quantitation (LLOQ) was 1 ng/mL for plasma and 5 ng/mL for breast milk. Assay methods were documented internally and calibrated to detect allopregnanolone levels during infusion conditions rather than detection of low endogenous levels. Pharmacokinetic parameters derived from plasma brexanolone concentrations by model-independent methods (non-compartmental analysis) included area under the concentration–time curve (AUC) from time 0–60 h (AUC0–60), AUC from time 0–infinity (AUC0–∞), AUC from time 24–56 h (AUC24–56) during the infusion plateau, maximum plasma drug concentration (Cmax), time to peak plasma concentration (tmax), steady-state drug concentration in plasma during constant-rate infusion (Css), clearance (CL), and average drug concentration in plasma at steady state during the infusion plateau (Cavg). Breast milk volume, allopregnanolone concentration (ng/mL), and amount of allopregnanolone (µg) were determined for each sample.

Safety was assessed by adverse events (AEs), vital signs, clinical laboratory measurements, electrocardiograms, and suicidal ideation and behavior (via Columbia Suicide Severity Rating Scale) [30]. All AEs were coded using the Medical Dictionary for Regulatory Activities Version 20.0. Treatment-emergent AEs (TEAEs; defined as AEs that occurred after treatment initiation) and serious AEs (SAEs) were recorded through day 7. Vital signs were measured at screening; pre-infusion; and at h 2, 4, 8, 12, 18, 24, 30, 36, 42, 48, 56, 60, 66, 72, and day 7. Clinical laboratory assessments were performed at screening, hour 72, and day 7. Electrocardiogram and C-SSRS were assessed at screening, pre-infusion, hour 72, and day 7.

Data from three randomized, double-blind, placebo-controlled trials (ClinicalTrials.gov identifiers: NCT02614547, NCT02942004, NCT02942017) [14, 15]; one open-label study (NCT02285504) [21]; and one open-label study of healthy lactating women (547-CLP-108; current study; see Electronic Supplementary Materials [ESM]) were used to evaluate the PopPK properties of brexanolone. Patients with PPD in the three controlled trials and healthy lactating women in the current study received a 60-h continuous intravenous infusion of brexanolone, with infusion rates escalating up either to BRX60 by hour 4 or to BRX90 by hour 24. Brexanolone was tapered during the last 8 h of the infusion. Four patients with PPD in the open-label study received a slightly different schedule of a single 60-h continuous infusion of BRX90 (see ESM). Plasma samples were collected for PK analyses at predefined time points and were assayed by an LC–MS assay with an LLOQ of 1 ng/mL.

A PopPK model describing brexanolone PK in plasma and breast milk was developed using the current study and supported by phase II/III data (N = 156; see ESM for details on study characteristics, PK sampling, and model development). PopPK modeling analyses were conducted using nonlinear mixed-effects modeling (NONMEM®, Version 7.3, ICON Development Solutions, Ellicott City, MD, USA; see ESM). Models of increasing complexity were examined with NONMEM and were diagnosed and qualified using criteria for graphical assessment of goodness-of-fit, visual predictive check, shrinkage, numerical assessment of goodness-of-fit, and assessment of uncertainty-of-parameter estimates. The general form of the model was a two-compartment model for plasma with estimation of a partition coefficient to account for the concentration gradient between plasma and breast milk. Body weight was included in the base PopPK model prior to stepwise covariate modeling. Later, with availability of data from the full set of clinical trials, the model for plasma PK was updated to incorporate all available results while keeping constant the estimates for milk obtained based on 547-CLP-108. See ESM for full details.

The safety set of the 547-CLP-108 study comprised all participants who were administered the study drug. The PK set comprised all participants with at least one evaluable PK sample. The breast milk set comprised all participants who began the infusion and had at least one breast milk sample collected. Continuous variables were summarized descriptively by number, mean, standard deviation, median, minimum, and maximum. Change from baseline values were calculated at each time point and summarized descriptively. Categorical variables were summarized with counts and percentages. Breast milk concentration of allopregnanolone, volume, and amount secreted were summarized by descriptive summary statistics, and are displayed in figures using both linear and log scales. Using the model parameters and assuming an infant feeding rate of 150 mL/kg/day [31], RID was computed as the infant dose divided by maternal dose, as previously described [32]. To test the hypothesis that concentrations in milk are a constant fraction of the plasma concentration and to support simulations in a more expansive population of patients, the model was used to characterize the shape of the milk concentration versus the time profile and to elucidate how long detectable levels of allopregnanolone might remain in the milk after cessation of dosing or at the end, after the completion of therapy.

$$\mathrm= \frac.\mathrm\left(\frac/\mathrm}}\right)}.\mathrm\left(\frac/\mathrm}}\right)},$$

where Dose.infant = dose in infant/day and Dose.mother = dose in mother/day.